FliW and FliS Function Independently To Control Cytoplasmic Flagellin Levels in Bacillus subtilis

The cytoplasmic level of flagellin (called Hag) is homeostatically regulated in the Gram-positive bacterium Bacillus subtilis by a partner-switching mechanism between the protein FliW and either the Hag structural protein or CsrA, an RNA binding protein that represses hag translation. Here we show that FliW and the putative secretion chaperone FliS bind to Hag simultaneously but control Hag translation by different mechanisms. While FliW directly inhibits CsrA activity, FliS antagonizes CsrA indirectly by binding to Hag, enhancing Hag secretion, and depleting Hag in the cytoplasm to trigger the FliW partner switch. Consistent with a role for FliS in potentiating Hag secretion, the mutation of fliS crippled both motility and flagellar filament assembly, and both phenotypes could be partially rescued by artificially increasing the concentration of the Hag substrate through the absence of CsrA. Furthermore, the absence of FliS resulted in an approximately 30-fold reduction in extracellular Hag accumulation in cells mutated for CsrA (to relieve homeostatic control) and the filament cap protein FliD (to secrete flagellin into the supernatant). Thus, we mechanistically discriminate between the FliW regulator and the FliS chaperone to show that secretion disrupts flagellin homeostasis and promotes high-level flagellin synthesis during the period of filament assembly in B. subtilis.     

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti‐σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far‐UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.     

Flagellin polymerisation control by a cytosolic export chaperone

Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments.     

Interaction of FliS flagellar chaperone with flagellin

Premature polymerization of flagellin (FliC), the main component of flagellar filaments, is prevented by the FliS chaperone in the cytosol. Interaction of FliS with flagellin was characterized by isothermal titration calorimetry producing an association constant of 1.9x10(7) M-1 and a binding stoichiometry of 1:1. Experiments with truncated FliC fragments demonstrated that the C-terminal disordered region of flagellin is essential for FliS binding. As revealed by thermal unfolding experiments, FliS does not function as an antifolding factor keeping flagellin in a secretion-competent conformation. Instead, FliS binding facilitates the formation of alpha-helical secondary structure in the chaperone binding region of flagellin.     

The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation

Assembly of each Salmonella typhimurium flagellum filament requires export and polymerisation of ca. 30000 flagellin (FliC) subunits. This is facilitated by the cytosolic chaperone FliS, which binds to the 494 residue FliC and inhibits its polymerisation. Yeast two-hybrid assays, co-purification and affinity blotting showed that FliS binds specifically to the C-terminal 40 amino acid component of the disordered D0 domain central to polymerisation. Without FliS binding, the C-terminus is degraded. Our data provide further support for the view that FliS is a domain-specific bodyguard preventing premature monomer interaction.     

The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin.

The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium.     

Flagellar filament elongation can be impaired by mutations in the hook protein FlgE of Salmonella typhimurium: a possible role of the hook as a passage for the anti-sigma factor FlgM

Among motile revertants isolated from flagellar hook-deficient ( flgE ) mutants of Salmonella typhimurium one produced only short flagellar filaments in L broth, despite the fact that flagellin itself has the ability to polymerize into long filaments in vitro . This pseudorevertant has an intragenic suppressor, resulting in a two-amino-acid substitution (Asp-Gln→Ala-Arg) in the C-terminal region of the hook protein, FlgE. The flagellation of the pseudorevertant was greatly affected by the concentration of NaCl in the culture media: we observed no filaments in the absence of NaCl, short filaments in 1% NaCl and full-length filaments in 2% NaCl. Electron microscopy of osmotically shocked cells showed that the number of hook–basal bodies on cells was constant under various NaCl conditions. Furthermore, we found that the mutant hook was straight rather than curved. We monitored the cellular flagellin level of this pseudorevertant under various NaCl concentrations by immunoblotting. It was revealed that little flagellin was present under NaCl-free conditions in contrast with the ordinary amounts of flagellin present in 2% NaCl. As the expression of flagellin is regulated by competitive interaction of a sigma factor, FliA, and a corresponding anti-sigma factor, FlgM, we also observed the effect of NaCl on the secretion of FlgM. FlgM was secreted into the media in more than 1% NaCl but accumulated inside the cells in the absence of NaCl, indicating that the failure of secretion of FlgM in the absence of salt was the cause of the impaired elongation of filaments.     

DegU-phosphate activates expression of the anti-sigma factor FlgM in Bacillus subtilis

The bacterial flagellum is a complex molecular machine that is assembled by more than 30 proteins and is rotated to propel cells either through liquids or over solid surfaces. Flagellar gene expression is extensively regulated to co-ordinate flagellar assembly in both space and time. In Bacillus subtilis, the proteins of unknown function, SwrA and SwrB, and the alternative sigma factor σ(D) are required to activate expression of the flagellar filament protein, flagellin. Here we determine that in the absence of SwrA and SwrB, the phosphorylated form of the response regulator DegU inhibits σ(D) -dependent gene expression indirectly by binding to the P(flgM) promoter region and activating expression of the anti-sigma factor FlgM. We further demonstrate that DegU-P-dependent activation of FlgM is essential to inhibit flagellin expression when flagellar basal body assembly is disrupted. Regulation of FlgM is poorly understood outside of Salmonella, and differential control of FlgM expression may be a common means of coupling flagellin expression to flagellar assembly.     

Interactions of bacterial flagellar chaperone–substrate complexes with FlhA contribute to co‐ordinating assembly of the flagellar filament

Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C‐terminal cytoplasmic domain of FlhA (FlhA_C), a membrane component of the export apparatus, provides a binding‐site for these chaperone–substrate complexes but it remains unknown how it co‐ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhA_C is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhA_C for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14‐fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhA_C for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.     

Maf‐dependent bacterial flagellin glycosylation occurs before chaperone binding and flagellar T3SS export

Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non‐glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form.     

Structural Basis for Stabilization of the Hypervariable D3 Domain of Salmonella Flagellin upon Filament Formation

The hypervariable D3 domain of Salmonella flagellin, composed of residues 190-283, is situated at the outer surface of flagellar filaments. A flagellin mutant deprived of the complete D3 domain (ΔD3_FliC) exhibited a significantly decreased thermal stability (T_m 41.9 °C) as compared to intact flagellin (T_m 47.3 °C). However, the stability of filaments formed from ΔD3_FliC subunits was virtually identical with that of native flagellar filaments. While D3 comprises the most stable part of monomeric flagellin playing an important role in the stabilization of the other two (D1 and D2) domains, the situation is reversed in the polymeric state. Upon filament formation, ordering of the disordered terminal regions of flagellin in the core part of the filament results in the stabilization of the radially arranged D1 and D2 domains, and there is a substantial increase of stability even in the distant outermost D3 domain, which is connected to D2 via a pair of short antiparallel β-strands. Our experiments revealed that crosslinking the ends of the isolated D3 domain through a disulfide bridge gives rise to a stabilization effect reminiscent of that observed upon polymerization. It appears that the short interdomain linker between domains D2 and D3 serves as a stabilization center that facilitates propagation of the conformational signal from the filament core to the outer part of filament. Because D3 is a largely independent part of flagellin, its replacement by heterologous proteins or domains might offer a promising approach for creation of various fusion proteins possessing polymerization ability.